The long-term goal of this proposal is to regenerate and repair dental pulp/dentin thereby reducing the need of endodontic procedures. Approximately 16 million teeth receive root canal treatment (~$14 billion) per year in the US-a procedure consisting of the removal of the entire pulp and replacing it with gutta percha (rubber-like material). There have been no other effective ways to repair the infected or injured pulp tissue besides complete amputation. Recent advances in pulp stem cell isolation and tissue engineering technologies have shed light on the possibility of pulp tissue regeneration. Removing the entire pulp poses two clinical problems: 1) further dental work such as crown and post that weaken the tooth are most often required; 2) devitalized immature teeth are weak with little dentin structure. Therefore the therapeutic capacity to regenerate pulp/dentin of the root would be clinically beneficial. Our preliminary data showed that human pulp-like structure constructed in vitro using stem cells and implanted in canals of human tooth fragments can be vascularized in the subcutaneous space of SCID mice. Therefore, we hypothesize that pulp-like tissue can be regenerated in tooth using stem cells aided with a scaffold system. In this proposal, we will characterize dental stem cells for regeneration of pulp/dentin and use mice and minipigs as ectopic and orthotopic animal study models respectively. There are three specific aims that stepwisely test the hypothesis to reach the goal: Aim 1- To characterize pulp stem cells of various sources for pulp regeneration. This aim will characterize prospective stem cells of various sources for pulp tissue regeneration. Human dental pulp stem cells (hDPSCs), human stem cells from apical papilla (hSCAP) and stem cells from human exfoliated deciduous teeth (SHED) will be isolated and characterized by examining their stem cell properties and gene expression profiles relevant to osteo/odontogenic potency using in vitro and in vivo experimentation. Procedures include flow cytometry, real-time RT-PCR, Western blot, immunohistochemical analysis, etc. Aim 2- To determine angiogenesis of human pulp-like tissues in vivo in SCID mice. The extent of angiogenesis is critical to the success of pulp regeneration. Stem cells seeded in D,L-lactide and glycolide (PLG) scaffolds containing angiogenic growth factors will be inserted into root canal space of human tooth fragments and implanted subcutaneously into SCID mice. The implanted teeth will then be harvested for histological analysis of cellularity, matrix production, formation of odontoblast-like cells and particularly vascularity. Gene expression profile of the regenerated tissue will be analyzed with and immunohistochemistry. Aim 3- To test pulp regeneration in a relevant tissue environment in swine. This experimental swine model simulates actual clinical applications in humans using autologous stem cells for pulp regeneration in pulp space that has been infected and then disinfected. Pulp stem cells from swine teeth will be seeded in scaffolds in vitro and planted back to the disinfected pulpless root canal space for de novo pulp regeneration or planted into pulp chamber space for partial pulp regeneration. The vascularization and innervations of the regenerated tissue will be determined with histology and immunohistochemistry. The success of this project would represent a potential breakthrough in clinical endodontics using stem cell therapy and serve as an example for other organ regeneration.